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J Dent Res 82(1): 23-27, 2003
© 2003 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Bone Morphogenetic Protein-2 Inhibits Differentiation and Mineralization of Cementoblasts in vitro

M. Zhao1, J.E. Berry1, and M.J. Somerman1,2,*

1 Department of Periodontics/Prevention/Geriatrics, University of Michigan School of Dentistry, 1011 N. University Avenue, Ann Arbor, MI 48109-1078, USA;
2 Department of Periodontics, School of Dentistry, University of Washington, D-322 Health Sciences Center, Box 356365, Seattle, WA 98195-6365, USA;

*corresponding author, somerman{at}u.washington.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
As an approach for improving the outcome and predictability of periodontal regenerative therapies, we have focused on determining the responses of cells within the local environment to putative regenerative factors. This study examined the effects of bone morphogenetic protein-2 (BMP-2) on murine cementoblasts in vitro. Northern blot analysis indicated that BMP-2 decreased mRNA levels of bone sialoprotein and type I collagen dose-dependently (10–300 ng/mL). At low doses, up to 100 ng/mL, BMP-2 had no effect on transcripts for osteocalcin and osteopontin, whereas at 300 ng/mL, BMP-2 greatly increased expression of these two genes. BMP-2 also inhibited cementoblast-mediated mineral nodule formation in a dose-dependent manner (inhibition was noted at 10 ng/mL). Noggin reversed the effects of BMP-2 on gene expression and on mineralization. These findings reflect the diverse responses of periodontal cells to BMP-2 and highlight the need to consider the complexity of factors involved in designing predictable regenerative therapies.

KEY WORDS: bone morphogenetic proteins • cementoblast • mineralization • periodontal regeneration • periodontal ligament


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cementum, a thin mineralized tissue covering the tooth root surface, assists in anchoring teeth to surrounding alveolar bone and thereby contributes to the maintenance of structural stability and physiological function of the dentition. Diseased cementum or defects in cementum formation are causal factors for structural disintegration and malfunction of the periodontium (Page and Baab, 1985). Therefore, considerable interest and attention have been targeted at clarifying specific factors, cells, and molecules involved in forming cementum, during both development and regeneration of periodontal tissues. In this regard, bone morphogenetic proteins (BMPs), known to enhance mineralization in many tissues, have emerged as attractive candidates for use in promoting the formation of periodontal tissues.

BMP proteins, belonging to the TGF-ß superfamily, have long been known as potent inducers of bone formation and also as factors involved in regulating tooth development (Wozney et al., 1988; Wang et al., 1990; Åberg et al., 1997; Helder et al., 1998). Results from animal studies suggest that BMPs have the ability to stimulate regeneration of periodontal tissues, including cementum (Sigurdsson et al., 1995; King et al., 1997; Ripamonti and Reddi, 1997; Giannobile et al., 1998), suggesting that BMPs may have clinical applications. However, in clinical trials, results of using BMPs, as well as other growth factors, have not been as promising as anticipated in regenerating periodontal tissues. There are most likely several factors contributing to this, including variability in patient responses, the types of delivery systems used, and a lack of understanding of the behavior of cells within the local environment (Bartold et al., 2000; Saygin et al., 2000).

In previous studies, we demonstrated that BMP-2 promoted dental follicle cells, putative progenitor cells for the periodontium, to differentiate into a cementoblastic/osteoblastic phenotype (Zhao et al., 2002). The objective of this present study was to determine the effects of BMP-2 on the mature cementoblasts, in vitro.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
All procedures were approved by the University Committee on Use and Care of Animals and were in compliance with state and Federal laws. In our laboratory, we established an immortalized murine cementoblast cell line, OC-CM, by isolating tooth root surface cells from transgenic mice containing a SV 40 large T-antigen (TAg) under the control of osteocalcin (OCN) promoter (Chen et al., 1995; D’Errico et al., 2000). OC-CM 30, a subclone of the immortalized OC-CM cell population, was used for all experiments. Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) plus 10% fetal bovine serum (FBS) containing 100 U/mL of penicillin and 100 µg/mL streptomycin (Gibco, BRL, Gaithersburg, MD, USA) in a humidified atmosphere of 5% CO2 at 37°C.

Cementoblast cell lines were established and selected based on our in situ hybridization data, i.e., only cementoblasts expressed strong BSP and OCN gene signals, while PDL cells did not (D’Errico et al., 1997). OC-CM 30 is considered a mature cell line because it expresses high levels of BSP and OCN transcripts, both of which are gene markers of mature osteoblasts/ cementoblasts. In contrast, precursor cell lines such as MC3T3-E1 cells and follicle cells require additional factors to initiate expression of BSP and OCN. In addition, the time selected for obtaining tissues for cell isolation was at a stage when root formation had already been initiated; thus, cells within the region are functioning as mature cementoblasts. Besides expressing BSP and OCN, OC-CM 30 cells also exhibit other phenotypic markers associated with cementoblasts, including cAMP up-regulation in response to PTH/PTHrP stimulation, and ability to form mineral nodules and mineralized tissue in an ex vivo SCID mice model (Somerman et al., 1999).

Northern Blot Analysis
Total RNA was isolated from cultured cells with the use of Trizol® reagent (Gibco BRL, Gaithersburg, MD, USA) and examined by Northern blot assay as previously described (Zhao et al., 2002). Blots were hybridized with random primed 32P-labeled probes (Rediprime, Amersham Pharmacia Biotech, Arlington Heights, IL, USA), recognizing mouse BSP (Young et al., 1994), mouse OCN (Celeste et al., 1986) (obtained from Dr. J. Wozney, Genetic Institute, Cambridge, MA, USA), bovine type I collagen (Col I), and mouse osteopontin (OPN) (Young et al., 1990). Hybridized blots were also scanned in a Packard A2024 Instantimager, and we normalized all values for RNA loading by probing blots with cDNA to 18S rRNA. Experiments were carried out three times with comparable results.

Western Blot Analysis
Western blot analysis was performed as described previously, for examination of the effects of BMP-2 on levels of BSP and OCN protein in culture media (Ouyang et al., 2000). After confluence was reached, cells were exposed to rhBMP-2 (0–300 ng/mL) (generous gift from Genetics Institute, Cambridge, MA, USA) with ascorbic acid (AA) in serum-free media for 48 hrs. Conditioned media (3 mL/treatment) were collected, lyophilized, and subsequently dissolved in 200 µL 1H SDS-PAGE loading buffer. A 10-µL aliquot of protein was fractionated by SDS-PAGE and transferred to PVDF membrane by means of a semi-dry transfer apparatus (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was probed with a primary rabbit antibody against mouse BSP (obtained from Dr. R.T. Franceschi; dilution 1:3000) or goat antibody against OCN (Biomedical Technologies Inc., Stoughton, MA, USA; dilution 1:500), followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (Promega, Madison, WI, USA) or rabbit anti-goat Ig G (Sigma, St. Louis, MO, USA) (1:10,000). The response was visualized with the use of an enhanced chemiluminescence (ECL) reagent, according to the manufacturer’s recommendation (Amersham Pharmacia Biotech). Experiments were performed three times with comparable results.

Assay of Mineral Nodule Formation
Cells were plated at the density of 4 x 104 cells/well in 24-well culture plates and cultured in media containing 10% FBS. Upon confluence (day 0), cells were cultured in mineralizing media (DMEM containing 5% FBS, 50 µg/mL AA, and 5 mM ß-glycerophosphate), supplemented with BMP-2 at different doses up to 300 ng/mL. Parallel cultures were treated with noggin (generously provided by Dr. Economides, Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA), a specific inhibitor of BMPs (Zimmerman et al., 1996), at the concentration of 1 µg/mL. Cell-mediated mineral nodule formation was determined on day 9 with von Kossa staining (Puchtler and Meloan, 1978). Experiments were performed three times with comparable results.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of BMP-2 on Gene Expression
Cells at confluence were exposed to BMP-2 (0–300 ng/mL), with or without AA, in DMEM containing 2% FBS, and media were changed every other day (Fig. 1Go). We extracted total RNA on day 5 to evaluate mRNA levels of BSP, OCN, Col I, and OPN, by Northern blot assay. BMP-2, in the presence of AA, reduced BSP and Col I mRNA in a dose-dependent manner. The decreased expression was noted at 10 ng/mL, and at 300 ng/mL the expression of these two genes was reduced by approximately 90% and 70%, respectively. BMP-2 had no effect on OCN and OPN expression at low doses up to 100 ng/mL, but at 300 ng/mL, BMP-2 up-regulated OCN and OPN mRNA expression by 2.5- and 3.5-fold, respectively. In the absence of AA, the effects of BMP-2 on gene expression were not as dramatic.



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  		Figure 1. Effect of BMP-2 on gene expression of cementoblasts. Cells were treated for 5 days with BMP-2 at concentrations up to 300 ng/mL, with or without AA, and Northern blot analyses were used to evaluate mineral-related gene markers. Results were normalized with 18S rRNA and are shown by bar graphs. Autoradiographs are representative of results obtained in three independent experiments.

 
Effect of Noggin on BMP-2-mediated Gene Response
We used noggin to identify the specificity of BMP-2 effect on gene expression (Fig. 2Go). After reaching confluence, cells were exposed to BMP-2 (300 ng/mL) or vehicle, with or without AA, for 48 hrs. Cells cultured in parallel were treated with noggin (1 µg/mL), and total RNA was extracted for the evaluation of BSP and OCN mRNA by Northern blot assay. BMP-2-mediated down-regulation of BSP and up-regulation of OCN mRNA levels were reversed by noggin, thus confirming the specificity of BMP-2 effects. Further, in the absence of BMP-2, noggin alone repressed the basal level of OCN transcript, but had no effect on BSP expression.



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  		Figure 2. Effect of noggin on BMP-2-mediated expression of BSP and OCN. Cells were cultured in DMEM with 2% FBS, ± BMP-2 (300 ng/mL), ± AA (50 µg/mL), ± noggin (1 µg/mL) for 5 days, and Northern blot analysis was used to determine gene expression of BSP and OCN. Results were normalized against 18S rRNA and are shown by bar graphs. Autoradiographs are representative of results obtained in three independent experiments.

 
Effect of BMP-2 on Protein Levelsof BSP and OCN
Conditioned media of cells treated with BMP-2 at different doses were collected and examined by Western Blot Assay for determination of the protein levels of BSP and OCN (Fig. 3Go). In agreement with its effect on BSP and OCN transcripts, BMP-2 at low doses (10–100 ng/mL) slightly reduced BSP protein and had no effect on OCN protein level. At the dose of 300 ng/mL, BMP-2 greatly reduced BSP protein and elevated OCN protein levels.



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  		Figure 3. Effect of BMP-2 on secretion of BSP and OCN protein. Cells at confluence were exposed to BMP-2 (0–300 ng/mL) with AA in serum-free media for 48 hrs. Conditioned media were collected and examined by Western blot assay with antibodies against BSP or OCN. This Fig. is representative of results obtained in three independent experiments.

 
Effect of BMP-2 on Mineral Nodule Formation
Consistent with its dose-dependent inhibition of BSP and Col I transcripts, two key molecules required for differentiation of cementoblasts and osteoblasts, BMP-2 inhibited cell-mediated mineral nodule formation in a dose-dependent fashion (Fig. 4Go). The inhibitory effect was observed at 10 ng/mL BMP-2. Noggin addition reversed the inhibitory effect of BMP-2 on mineralization, while noggin alone had no effect on mineralization.



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  		Figure 4. Effect of BMP-2 on cell-mediated mineral nodule formation. Cementoblasts were cultured in mineralizing media supplemented with BMP-2 (0–300 ng/mL) with or without noggin (1 µg/mL). To evaluate mineral nodule formation, we performed von Kossa staining on day 9.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Results from the present study suggest that BMP-2 has the capacity to regulate cementoblast behavior, e.g., expression of genes associated with mineralization; however, the outcomes of the study were not as anticipated. In contrast to our prediction that BMP-2 would enhance expression of genes associated with mineralization and, subsequently, mineral formation, exposure of cementoblasts to BMP-2 resulted in dose-dependent reduction of BSP and Col I gene expression, and inhibition of cell-induced mineral nodule formation. These effects were not linked to cell proliferation, since exposure of OC-CM cells to BMP-2 did not change cell growth in comparison with control cells (data not shown). Although BMP-2 induced the inhibition of mineralization, alkaline phosphatase activity was not repressed in cementoblasts exposed to BMP-2 (results not shown). At this point, the most logical explanation for BMP-2’s inhibitory effects on cementoblast-mediated mineral nodule formation is the down-regulation of BSP and Col I, at both gene and protein levels. This is consistent with the evidence that BSP plays a key role in the initiation of mineral formation (Hunter and Goldberg, 1993), and that collagen within the extracellular matrix is essential for promoting osteoblastic differentiation (Xiao et al., 1998; Jikko et al., 1999). BMP-2-induced increase in OCN and OPN gene expression does not appear to play a major role in the inhibition of mineral nodule formation, since these effects were noticed at only a high concentration (300 ng/mL), whereas the inhibitory effect on mineralization was seen dose-dependently, from 10 to 300 ng/mL of BMP-2.

Experiments with noggin, a specific inhibitor of BMPs, confirmed the specificity of BMP-2 action on both gene expression and mineral nodule formation. Further, the fact that noggin alone decreased OCN expression but had no effect on BSP expression suggests that endogenous BMPs are important in maintaining levels of OCN mRNA. This finding, as well as the fact that transcripts for BSP and OCN were differentially regulated by BMP-2, suggests that BSP and OCN genes are controlled by separate signaling pathways and/or transcription factors, or, alternatively, by the same transcription factors with different effects on their respective promoters. The transcription factor Cbfa1 (core-binding factor alpha 1) has been shown to be a key regulator of osteogenic differentiation (Ducy et al., 1997; Komori et al., 1997). BMP-2 has been reported to increase Cbfa1 mRNA levels in several immortalized cell lines, including human bone marrow stromal cell line (Gori et al., 1999), C2C12 cells (Lee et al., 1999), and 2T3 cells (Chen et al., 1998), and to promote these cells to differentiate along the osteoblastic pathways. Multiple Cbfa1 binding sites have been identified in the OCN promoter, and the positive regulatory effect of Cbfa1 on OCN gene expression has been confirmed (Banerjee et al., 1996; Ducy et al., 1997; Javed et al., 1999). In contrast, while Cbfa1 binding sites are present in the BSP promoter region, the effects of Cbfa1 on BSP gene regulation remain unclear and contradictory (Ducy et al., 1997; Benson et al., 1999; Javed et al., 2001; Xiao et al., 2002). Some studies suggest that the ability of BMPs to increase Cbfa1 mRNA levels correlates with increased BSP gene transcripts (Ducy et al., 1997; Xiao et al., 2002), while analysis of other data suggests that Cbfa1 sites in the murine BSP promoter lack enhancer activity (Benson et al., 1999), and another study shows that Cbfa1 mediates repression of Gallus BSP promoter activity in both osteogenic and non-osteogenic cells (Javed et al., 2001). Considering the possibility that the differential regulation of BSP and OCN genes by BMP-2 in the present study might be in accordance with the findings described above, we examined the effects of BMP-2 on Cbfa1 mRNA levels in OC-CM cells. Although BMP-2 had no effect on Cbfa1 transcripts (data not shown), it remains to be determined whether or not BMP-2 treatment enhanced the DNA binding ability of Cbfa1 through post-translational modification such as phosphorylation, as reported for murine osteoprogenitor MC3T3-E1 cells (Xiao et al., 2000). Detailed investigations on the signaling pathways involved in BMP-2-induced changes in gene expression in cementoblasts are ongoing in our laboratory.

There is general consensus that BMPs play a promotive role in the process of osteoblastic differentiation, especially in the early, maturing stage, although their function in mature osteoblasts is less clear. Recently, a unique kinase named BIKe (BMP-2-Inducible-Kinase) was identified and cloned in a BMP-2-treated pre-chondroblastic cell line, MLB13MYC clone 17 (Kearns et al., 2001). This kinase was also found to be increased in mature MC3T3-E1 cells during prolonged culture. When stably expressed in MC3T3-E1 cells, BIKe significantly decreased alkaline phosphatase activity and OCN mRNA levels and retarded in vitro mineral deposition. This finding provides another regulatory mechanism that attenuates the differentiation and maturation of osteoblastic cells. Thus, it seems plausible that the effects of BMP-2 treatment reported in our present study are not unique to mature cementoblasts, but rather are common to mature mineral-inducing cells in vitro. Importantly, we have shown that when follicle cells, the putative precursor cells for cementoblasts, are exposed to BMP-2, they are induced to express transcripts for BSP and OCN, and also to mineralize in culture (Zhao et al., 2002). Similarly, other researchers have demonstrated the promotion of osteoblastic differentiation of progenitor cells upon exposure to BMP-2 (Chen et al., 1998; Gori et al., 1999; Lee et al., 1999).

Other support for our findings comes from studies by Rajshankar et al. (1998). Using a periodontal wound model in rats, they reported that high doses (2.5 µg/mL) of BMP-7 (similar to BMP-2 in effect) applied to the PDL region failed to alter the PDL width or to promote ankylosis. They concluded that the PDL region might contain anti-osteogenic cell populations involved in the preservation of PDL width. Given the response of mature cementoblasts to BMP-2 shown in the present study, factors secreted by cells lining the root surface in the fully developed periodontium may also act as regulators of crystal growth. Previously, we reported that BMP-2 enhances differentiation of dental follicle cells along a cementoblastic pathway (Zhao et al., 2002). Therefore, the reported positive effect of BMP-2 in promoting mineralization is most likely related to its interactions with progenitor cells within the PDL region and from marrow spaces. In contrast, the more mature cells in the environment, e.g., cementoblasts, may react to BMP-2 in a different way, to prevent ankylosis.

To the best of our knowledge, this is the first in vitro study directed toward determining the effects of BMP proteins on cloned cementoblastic cells. Although the in vitro findings may not necessarily represent the actual events in vivo, they reflect the diverse responses of cells within the periodontium to specific factors and highlight the need to consider the complexity of factors involved in designing regenerative therapies.


   ACKNOWLEDGMENTS
 
This investigative study was supported by USPHS Research Grants DE-013047 and DE-09532 from the National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892. We thank Genetics Institute (Cambridge, MA, USA) for providing rhBMP-2 protein and Dr. Economides, Regeneron Pharmaceuticals, Inc. (Tarrytown, NY, USA), for providing noggin protein. This study was presented at the 80th General Session of the IADR, March 6–9, 2002, San Diego, CA, USA (J Dent Res 81[Spec Iss A]:A505, #4136).

Received May 6, 2002; Last revision August 12, 2002; Accepted October 2, 2002


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 MATERIALS & METHODS
 RESULTS
 DISCUSSION
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